Modelling rare disease in Xenopus laevis using CRISPR/Cas9 (689)

Meier-Gorlin syndrome is a rare autosomal recessive disorder characterized by a triad of features: short stature, microtia and absent patellae. Patients can also display a range of skeletal abnormalities. Five genes causative genes involved the formation of the pre-replication complex have been identified namely: ORC1, ORC4, ORC6, CDT1 and CDC6 as well as CDC45, a protein involved in the pre-initiation complex.

Skeletal development is conserved between frogs and humans. We aimed to investigate the skeletal features of this disease using cutting edge CRISPR/Cas9 technology to disrupted orc1 in X. laevis.

Methods

gRNA were designed to target X.orc1, these were microinjected into one cell stage embryos along with Cas9 mRNA. We also designed Antisense morpholino oligios against orc1 which inhibit translation of orc1 mRNA into protein these were microinjected into two cell embryos.

Results

X.orc1 edited tadpoles display skeletal abnormalities reminiscent of Meier-Gorlin syndrome including delayed bone age and movement deformities at the knee joint. X.orc1 edited tadpoles are developmentally delayed and smaller in size when compared with control siblings at late tadpole stages. Surprisingly they do not display obvious growth phenotype early in development.

Antisense morpholino oligios were used in to attempt to recapitulate the early growth phenotype seen in Meier-Gorlin patients. Morpholino oligio treated embryos show demonstrable reduction in growth when compared to control siblings.

conclusions

These results show proof of concept that CRISPR/Cas9 edited Xenopus frogs have potential to act as models for rare human diseases.